LSD1 inhibition circumvents glucocorticoid-induced muscle wasting of male mice

Synthetic glucocorticoids (GC), such as dexamethasone, are extensively used to treat chronic inflammation and autoimmune disorders. However, long-term treatments are limited by various side effects, including muscle atrophy. GC activities are mediated by the glucocorticoid receptor (GR), that regulates target gene expression in various tissues in association with cell-specific co-regulators. Here we show that GR and the lysine-specific demethylase 1 (LSD1) interact in myofibers of male mice, and that LSD1 connects GR-bound enhancers with NRF1-associated promoters to stimulate target gene expression. In addition, we unravel that LSD1 demethylase activity is required for triggering starvation- and dexamethasone-induced skeletal muscle proteolysis in collaboration with GR. Importantly, inhibition of LSD1 circumvents muscle wasting induced by pharmacological levels of dexamethasone, without affecting their anti-inflammatory activities. Thus, our findings provide mechanistic insights into the muscle-specific GC activities, and highlight the therapeutic potential of targeting GR co-regulators to limit corticotherapy-induced side effects.

the authors may need to validate the muscle atrophy by examining the muscle fiber type upon dexamethasone treatment.8.It is unclear whether LSD1 has a cell-autonomous role in skeletal muscle.The authors may perform in vitro experiments involving knock-down and overexpression of LSD1 using C2C12 or murine primary myotubes to rule out any possibility of paracrine and autocrine signaling influencing the muscle phenotypes in the mice models used for this study.9. Numerous critical concerns exist with the immunoprecipitation experiments in the paper: The authors have used 10 % input, and most of the immunoprecipitated lanes have stronger bands than the input lane, which is inappropriate and impossible.Also, the authors have used single IgG control for two independent immunoprecipitation reactions performed with two different antibodies, which may also be wrong.Fig. 1B, 2D, 2F, 3A, 5F: It is unexpected to observe greater expression of the interacting partner protein than the immunoprecipitated protein.Please confirm if the origin of the secondary antibodies against LSD1 and GR primary antibodies is the same.Please verify the results and add loading controls to the panels.Note that, oddly, the input bands in the top and bottom panels are non-identical in Fig. 1B.In Fig. 4G: Are the IgG antibodies used for Trim63 and Ddit4 of different origins?10.Critical controls are missing in the figures.In Figure 6, (A-E), extended figures 6 and 7B-the CC-90011 alone treatment group is missing.Without this group, it would be hard to interpret the data shown in Figures 6, extended Figures 6 and 7. 11.Fig. 1G-I: Given that GR is a major transcription factor for atrogenes, it is crucial to reveal whether LSD1 interacts with the atrogene loci in myofibres at physiological GC levels.12. Fig. 6A, B: It is difficult to interpret the effect of LSD on DEXA-induced muscle atrophy without first ascertaining the impact of the LSD1 inhibitor itself on GR activity.It is strongly recommended to validate the inhibitor in the mouse model used.13.Since the primary outcome of the paper is to characterize the use of LSD1 inhibitor to circumvent the muscle atrophy phenotype associated with the use of DEXA as an anti-inflammatory drug, it is crucial to observe the cytokine and chemokine profile in DEX-treated control and LSD-/-mice, in addition to the immune cell population shown in the manuscript.14.Fig. S1H-I: Please assess the extent and tissue specificity of LSD1 depletion in the mouse model by western blotting.The authors may test the expression of LSD1 in different muscles and organs.15.Fig. 3: To assess the role of the GR/LSD1 complex in muscle atrophy, it is imperative to evaluate the activity of GR under starvation conditions in control and LSDskm-/-mice.16.Fig. S4F: Given that FAD is depletion upon starvation, the enzymatic activity of LSD1 may be expected to reduce upon food deprivation.It is, therefore, important to investigate and comment upon the timesensitive role of LSD1 in GR-mediated activation of starvation-induced muscle atrophy.17.Fig. S1J, 3A, 3E, 4C: Throughout the paper, authors have characterized LSD function almost exclusively in the gastrocnemius muscle.Verifying these critical results in other important muscle types, especially the tibialis anterior (among other Type I and Type II muscle fibers), would be interesting.18. Fig. 3E: Electron microscopy images are required to be quantified.Also, these results need to be verified by other methods.19.Fig. 5D, E: Inconsistent use of loading controls in western blotting.20.Fig. 4C, 5D-E: The authors have not quantified the western blots in this manuscript.All western blots need to be quantified and presented.21.Extended figure 3C-fiber cross-section area (CSA) of GR(i)skm-/-mice starved for 48 h is much higher than the GR(i)skm-/-mice fed animals, which I feel is impossible.Fig. S3C: This figure indicates that starvation induces hypertrophy in the muscles of GR-/-mice.This is highly improbable since starvation is well known to inhibit protein synthesis.Furthermore, Fig. S3H indicates upregulation in mRNA expression of atrogenes, contradicting the hypertrophy phenotype reported.Minor comments: 1. Please ensure uniform color coding across data figures in the manuscript.2. Fig. 1H, S1E: Since the study is focused on skeletal muscles, please avoid displaying cardiac-associated genes in the list to avoid confusion.3. Fig. 6, 7B: Please denote groups as Vehicle, DEX, and DEX+CC in the figure for simplicity.
Reviewer #2 (Remarks to the Author): In this manuscript, Cai and colleagues investigate the molecular details underlying glucocorticoidinduced skeletal muscle atrophy, which limits the use of synthetic GC in patients that require chronic administrations.They report that LSD1 interacts with both GR and NRF1 to regulate gene expression.Pharmacological inhibitors of LSD1 attenuate muscle atrophy, providing evidence for the involvement of LSD1 and therapeutic promising applications.The number of reports that establish a role for LSD1 in skeletal muscle is increasing, so is the tight relationship with steroid hormone receptors.The manuscript is very well written, results are clear and conclusions are appropriate.Before publication, we have the following suggestions that may improve to strength the authors' conclusions.1) In Fig. 1 the authors show colocalization of LSD1 and GR but not all the nuclei are positive, can the author stain for the different fiber types with MyHC marker subtype to establish whether there is a preferential expression of type IIb vs intermediate or oxidative fibers?Is there a homogeneous distribution of double positive nuclei in the entire transversal section, as the fiber type composition of gastrocnemius is not uniform.2) In the IP experiment, the lower band of LSD1, that also the authors notice, is interesting, could that be another isoform (alternative splicing) or another member of the KDM family that interacts with GR? IP with an anti-GR Ab pulls down a lower band that is not visible in the input, may be because it is not enriched enough, but the upper isoform is not visible.This is problematic.However, CHIP analysis shows interaction at the chromatin level.But the authors should clarify why the bands have different size, or reconsider the interpretation of the IP experiments.LSD1 has isoforms (2a, 8a, etc).
3) Were the CHIP on target validations performed on different samples as those used for CHIPseq?If so, this strengthens the validation analysis.4) The first sentence of the figure captions may be more informative with respect to "Characterization of…", "Role of…" can it be a sentence summarizing the main message of the figure?5) Is the interaction of GR with LSD1 and NRF1 modulated by ligand?Can this be tested in cells treated with vehicle or GC? 6) The observation that LSD1 is required for interaction of GR with NRF1 (Fig 2F) suggests a specific effect, is it possible that LSD1 demethylates GR? Or is this effect (fig 2G) occurring at the chromatin level?7) I suggest to move to main figure 3 analysis of CSA in control and starved mice with the relative quantification.Relative to autophagy, WB analysis of p62 and LC3 I/II should be added to conclude that autophagy is not induced and to determine is the autophagy flux is regular.
8) What would be the benefit to use CC-90011 with respect to TCP, for instance?9) The effect of the inhibitor (occurring through the catalytic activity) implies that LSD1 demethylase activity is important, is it only involved in histone modification or is it the result of direct modification of GR or NRF1? 10) And what is the involvement of NRF1 in muscle atrophy?What do the data shown in Fig 2 add to the rest of the story?11) The discussion and the manuscript would benefit if the authors take into consideration recent findings that establish a role of LSD1 in muscle in the cotext of another steroid receptor, androgen receptor.Especially considering the anabolic effects of androgen signaling in muscle.LSD1 represses transcription of several genes, but it transactivates AR, is this the case for GR? Similar specific effect that boosts GR transactivation?12) For statistical analysis, ANOVa (not t test) shall be used for more than 2 sample comparisons.
Reviewer #3 (Remarks to the Author): The study by Cai and collaborators investigates the relationship between the lysine-specific demethylase LSD1 and the glucocorticoid receptor (GR) in the context of muscle atrophy.The Authors show that these molecules directly interact resulting in the modulation of target gene expression.Among others, gene involved in the regulation of muscle mass homeostasis are targeted by LSD1 and GR interaction.The study is mainly based on genetic approaches, also taking advantage of mice in which the expression of GR or LSD1 is down-regulated.The results showing the interaction bewteen LSD1 and GR are quite convincing, while few notes of care arise as for the relevance of such interaction to muscle atrophy, which is investigated in mice exposed to fasting or administered dexamethasone (DEX).Specifically, the following issues should be taken into consideration: • line 85-86: while it is conceivable that some GRs are located in the nucleus also in physiological conditions such as those analyzed by the Authors, Figure 1A shows no or very little cytoplasmic GR, while this latter should be its main location in this experimental setting; • one of the main findings reported in the present study is that muscle-specific lack of LSD1 confers a significant resistance to wasting induced in mice (age?initial body weight?) by 48 h fasting.Such a pattern is associated with inhibition of the expression of genes involved in catabolic pathways such as muscle-specific ubiquitin ligases and autophagy and with preservation of muscle ultrastructure.However, these results do not rule out that other pathways in addition to the GR-dependent one(s) could be altered by the lack of LSD1 in the skeletal muscle.In this regard, 48 h fasting results in a severe substrate shortage.While adipose tissue reduction, more marked in LDS1-/-mice than in controls, likely attempts to compensate such a shortage, it is hardly able to achieve a total 'buffer' against the lack of nutrients; • it would have been interesting to see the effects of LSD1 inhibitor in fasted animals; • the second main observation of the study is that LSD1 down-regulation protects mice against DEXinduced atrophy, apparently without affecting DEX anti-inflammatory properties.However, only indirect evidence is provided in this regard, showing that spleen changes in size and both spleen resident and circulating immune cell sub-populations do not differ in the presence or in the absence of the LSD1 inhibitor.In this regard, the lack of effect on the anti-inflammatory properties should be demonstrated using the LSD1 inhibitor on models such as the experimental autoimmune encephalomyelitis or inflammatory bowel disease; • control mice treated with the LSD1 inhibitor are not included in the experimental design, while they should; • LSD1 lack of action on the anti-inflammatory activity does not really fit with data reported in Figure 1I, which shows an up-regulation of genes involved in the Inflammatory Response Pathway; • most of the results obtained in starved or DEX-exposed mice refer to gene expression data.However, since changes of mRNA levels do not necessarily match with protein expression and activity, at least some of the markers of protein hypercatabolism should be assessed at the protein level; • data on human myotubes do not add any particular cue, they could be cited as supplemental; • it not correct to talk about 'muscle catabolism' or 'muscle degradation'.Indeed,a tissue per se cannot be degraded or synthetized, rather its components (protein, lipids, etc.) are processed during anabolic/catabolic reactions.Similarly, it is not correct to talk about muscle atrophy when referring to an in vitro system (line 56); • finally, in lines 203-204 LSD1 is reported to play a role in 'initiating the food deprivation process', which is a non-sense, since such a process starts when mice are no more allowed to reach food.

Reviewer #1 (Remarks to the Author):
In this study, the authors outline a regulatory role for LSD1 as a co-ac9vator of the glucocor9coid receptor (GR) in skeletal muscles.Using LSD1-/-and GR-/-mice models, they claim that LSD1 coordinates the interac9on between GR and its enhancer protein, NRF1, to s9mulate the downstream expression of genes under basal condi9ons.Further, they also state that LSD1 promotes GR-mediated atrogene expression upon starva9on.Finally, they demonstrate that the GR-LSD1 complex may mediate dexamethasone-induced muscle atrophy in mice.There is considerable interest in therapeu9c-based approaches to prevent muscle was9ng.Thus, characterizing the role of a poten9al therapeu9c target, namely LSD1, in DEX-mediated muscle atrophy would greatly interest the wider research community.Although the premise of the study is promising, there are various cri9cal concerns, especially regarding the issues in the experimenta9on, data analysis, and lack of clarity on the mechanism.We thank the reviewer for her/his 9me and exper9se in reviewing our submission, and are grateful for her/his construc9ve feedback.Note however that number of concerns were already discussed in the original version of the manuscript (see below).
Major comments: 1.I have concerns about the novelty of the manuscript.The role of LSD1 is demonstrated in the muscles (PMID: 20833138, PMID: 28228264, PMID: 36695573, and PMID: 29371665).A recent study also showed that the skeletal muscle-specific LSD1 loss exacerbated glucocor9coid-induced atrophy (Araki et al., eLife 2023), which is in contrast to the findings of this work.It is well-accepted that contras9ng results are possible, but the author never a^empted to explain the differences in the phenotype and the mechanisms found in this study.In response to the concerns pertaining to the novelty of our findings, LSD1's role has been mainly studied in myogenesis (PMID: 20833138: Choi et al 2010 role of LSD1 in C2C12 myoblast differen9a9on, PMID: 28228264: Scion9 et al. 2017 role of LSD1 in early muscle differen9a9on, and PMID: 29371665: Tosic et al.Lsd1 regulates skeletal muscle regenera9on and directs the fate of satellite cells).While in the study of Araki et al. (2023, PMID: 36695573) LSD1 defines the fiber type-selec9ve responsiveness to environmental stress by considering its interac9on with FOXK1 in fast fibers and with ERRg in slow fibers, our manuscript offers unique findings in several ways: 1) beyond emphasizing LSD1's func9on as a GR co-ac9vator, we dissect its intricate molecular mechanism in orchestra9ng glucocor9coidinduced muscular atrophy, 2) our genome-wide analyses provide comprehensive evidence of LSD1's func9onal importance, mapping out about 16,000 LSD1 binding sites that cooccur with those of GR at enhancers and those of NRF1 at promoters, 3) we have detailed how LSD1 is required for GR-mediated muscle atrophy in starva9on and pharmacological condi9ons, which provides a broader insight into the mul9faceted roles of LSD1 in skeletal muscles.As men9oned in the original version of the discussion discussion (page 14, lines 314-322, now page 18, lines 420-432), we provide possible explana9ons for the discrepancies with the study of Araki and colleagues (PMID: 36695573).Indeed, the mouse models used in both studies differ substan9ally.Our model was generated by dele9ng the exon 1 of Lsd1, leading to an early frame shie that prevents protein transla9on.In contrast, Araki et al. inves9gated LSD1 mutants in which exons 5 and 6 were deleted, which might result in a C-terminal truncated protein lacking the MAO domain and the TOWER domains.Since the authors used an an9body directed against LSD1 C-terminal domain, LSD1 truncated protein could not have been detected but might have had dominant nega9ve effects, especially given the pivotal role of the N-terminal SWIRM domain in facilita9ng interac9ons with transcrip9on factors and ancillary co-factors.Moreover, the 9me and length of TAM and DEX treatment also differ with the study of Araki and colleagues, in which DEX was administered concomitantly with TAM, thereby causing two main issues: 1) LSD1 might have been not fully depleted when DEX was applied, thus contribu9ng to the promo9on of atrogene transcrip9on, and 2) altered estrogen receptor ac9vity might interfere with that of GR when their ligand are applied simultaneously.In contrast, in our study, DEX was administered one-month aeer TAM injec9on.In addi9on, we show that both LSD1 abla9on in skeletal muscle and LSD1 pharmacological inhibi9on counteracts DEX-induced muscle atrophy.
Altogether, we believe that our findings significantly enhance the knowledge on LSD1's func9on in skeletal muscles, and provide a strong founda9on for future research to improve glucocor9coid treatments by minimizing muscle atrophy.
2. The authors show that LSD1 is required for the interac9on between GR and its enhancer protein, NRF1, to s9mulate the downstream expression of genes under basal condi9ons.However, it is unclear how LSD1 may regulate NRF-1 to mediate GR ac9vity in muscles.It would be worthwhile to explore the role of NRF1-dependent regula9on of GR by LSD1 to gain a be^er mechanis9c insight into the process.In response to this concern, our RNA-seq analyses revealed that LSD1 loss in myofibers does not affect Nrf1 and GR transcript levels.Moreover, as we stated in the discussion (page 14, lines 277-285, now page 16, lines 377-385), we demonstrate that LSD1 directly interacts with GR at enhancer regions and with NRF1 at promoters, and that these interac9ons are required to connect GR and NRF1, and s9mulate gene expression by demethyla9ng the H3K9me2 repressive mark.Indeed, our co-IP (Fig. 2f) and ChIP-qPCR experiments (Fig. 2g) demonstrate that LSD1 is essen9al for the op9mal interac9on between GR and NRF1 under basal condi9ons to promote target gene expression.These coopera9ve interac9ons establish a func9onal link between GR at enhancer regions and the transcrip9on factor NRF1 at promoter sites in myofibers.In addi9on, our previous study revealed that GR recruitment to target genes was decreased by 50 % upon Nrf1 knock-down, and that Nrf1 silencing decreased the transcript levels of the GR target genes Eif4ebp2 and Pik3r1 by 50 % without impac9ng GR expression (doi: 10.1093/nar/gkab226), thereby showing that NRF1 is required for GR-dependent transcrip9on.These datasets are further supported by our new dataset showing that GR, LSD1 and NRF1 silencing decreases the expression of GR target genes (new Fig. 2m and S2i).1C-E, the authors examine the GR and LSD1 genomic localiza9on in myofibers and find that 80% of the DNA segments bound by LSD1 were also bound by GR.Are these DNA segments bound by NRF1 as well?This needs to be demonstrated as the manuscript also aimed to characterize the NRF1.To address this point, we have conducted a ChIP-seq analysis on NRF1 (new Fig. 2h-l and S2e-g).In agreement with our hypothesis, our studies reveal that the presence of GR and LSD1 at promoters strongly correlates with that of NRF1, in contrast with GR/LSD1 bound enhancers where NRF1 signal was not detectable (new Fig. 2i, j and S2g).These data were validated by independent ChIP-qPCR experiments at various loci (Fig. 2g and new S2h).These results further strengthen our understanding of the tripar9te interac9ons at the genomic level (pages 7-8 lines 167-182).

In Fig
4. Previous studies have demonstrated that LSD1 regulates mitochondrial oxida9ve phosphoryla9on and fa^y acid metabolism in muscles.I am wondering about the status of these genes in the model used in this study.Did the muscle-specific and whole-body LDS1-/-mice exhibit changes in metabolism, especially in oxida9ve phosphoryla9on and fa^y acid metabolism in muscles?To our knowledge, there is no study describing LSD1 role on mitochondrial oxida9ve phosphoryla9on and fa^y acid metabolism in muscles of adult mice.This topic has mainly been addressed in either a developmental context, in fat 9ssue or in cancer cells (doi: 10.1182/bloodadvances.2020003521, 10.1038/ncomms5093, 10.1016/j.celrep.2016.09.053), which was addressed in the original version of the discussion (page 13, lines 286-292, now page 17, lines 388-394).In this manuscript we analyzed either cons9tu9ve (LSD1 skm-/-) or TAM-inducible (LSD1 (i)skm-/-) musclespecific LSD1 knock-out mice, as whole-body LSD1-/-mice die during embryonic development (h^ps://doi.org/10.1038/ng.268,doi: 10.1101/gad.2008511,doi: 10.1128/MCB.00521-10,doi: 10.1038/nature05671).The analysis of our RNA-seq data reveals that LSD1 abla9on in myofibers does not affect the expression of genes involved in mitochondrial oxida9ve phosphoryla9on and has no major influence on fa^y acid metabolism-related genes (Fig. R1), in contrast to what we previously described in the fat 9ssue where LSD1 control the vast majority of genes involved in OXPHOS and oxida9ve metabolism (DOI: 10.1038/ncomms5093).Instead, our data reveal that LSD1 is rather associated with immune response (Fig. R2).Addi9onal pathway analysis of LSD1 direct targets is now presented in new Fig.S1n. 5.The authors have used starva9on as a model to study whether LSD1 controls GR transcrip9onal ac9vity.However, no data is shown whether the GR signaling is ac9vated in this model.How were the authors convinced GR signaling is involved in this model, as starva9on is known to induce mul9ple signaling pathways independent of GR in the muscle cells to induce atrophy?
6.The authors have used tamoxifen-dependent CreERT2 system-generated LSD1(i)skm-/-mice.Studies have shown that tamoxifen induces toxicity in muscles.How were the DEX injec9on experiments performed post-tamoxifen injec9on?The methods are unclear in the manuscript.It was shown that long-term treatment of breath cancer with tamoxifen can induce myalgia, muscle aches and cramps in rare cases.No other signs of toxicity have been reported at recommended doses to our knowledge (doi:10.1007/s00280-014-2605-7).In our model, mice are treated for 5 consecu9ve days with Tamoxifen to induce CreER T2 transloca9on to the nucleus, and DEX treatments were performed one month aeer TAM injec9ons (page 18, line 426).Previous studies from our group showed that tamoxifen levels are neglectable already 3 days aeer the last injec9on since the CreER T2 expression was mainly cytoplasma9c at this 9me (h^ps://doi.org/10.1006/meth.2001.1159).Note that control mice were also treated with TAM and do not present histological defects (new Fig. S3c), thus preven9ng a poten9al bias caused by this compound.
7. Muscle-specific and whole-body LDS1-/-mice are the animal models used in this study.However, this mouse is not characterized well.It is essen9al to characterize the mouse model for various physiological parameters, including age-wise characteriza9on of body weight, food, and water intake, blood profiling, etc.Also, characterize overall muscle phenotype in these mice under basal condi9ons by performing treadmill run experiments, metabolic cage experiments, and SDS COX staining at the least.As men9oned in point 4, we did not use whole-body LSD1-/-mice since it is lethal, but muscle-specific LSD1 knock-out mice.Since this manuscript focuses on the role of LSD1 in GC-induced muscle atrophy, we believe that characterizing the role of LSD1 in metabolism is out the scope of this study.Our data nevertheless show that muscle histology of mice with myofiber-specific LSD1 abla9on does not lead to gross abnormali9es (page 5 lines 116-119, Fig. S1k).In addi9on, body, muscle and WAT weight, as well as muscle strength are displayed in new Fig.S1l, m, and are similar between control and LSD1 skm-/-mice.Muscle weight and strength, as well as transcript levels of well-known atrogenes (Atrogin/Fbxo32 and Murf1/Trim63) were determined to validate muscle atrophy (new Fig. 5a-c, S5a-c and 7a-c).
In this work, the authors may need to validate the muscle atrophy by examining the muscle fiber type upon dexamethasone treatment.It is well-known that eleva9on of GC levels leads mainly to type 2B myofiber atrophy (h^ps://doi.org/10.1016/j.cmet.2011.01.001,DOI: 10.1152/jappl.1995.78.2.629, h^p://dx.doi.org/10.1016/j.mce.2013.03.003).In light to these data, we show that fast-twitch muscles such as gastrocnemius and quadriceps are the most affected by DEX treatment.We now provide evidence that soleus muscle weight is not altered by GCs (Fig. 5a, S5a and 7a).A sentence explaining the differences between slow-and fast-twitch muscles' DEX response has been added in the discussion (page 17 lines 399-402).
8. It is unclear whether LSD1 has a cell-autonomous role in skeletal muscle.The authors may perform in vitro experiments involving knock-down and overexpression of LSD1 using C2C12 or murine primary myotubes to rule out any possibility of paracrine and autocrine signaling influencing the muscle phenotypes in the mice models used for this study.To demonstrate that LSD1's func9on in skeletal muscle is cell-autonomous, we engineered and analyzed two muscle-specific mouse models of LSD1 abla9on.We demonstrate that LSD1 binding at DNA in muscle is abolished in LSD1 (i)skm-/-mice, thereby showing its myofiber specificity.In addi9on, we used in the original version of the manuscript the LHCNM2 9ssue culture model of human myofibers to test the GR-LSD1 func9onal interac9on in the presence or absence of ligand (former Fig. 7, new Fig. 6).We now provide further evidence that GR/LSD1/NRF1 target gene expression is decreased upon silencing (new Fig. 2m and S2i).Thus, while we acknowledge that in vitro models offer more direct prove of cell-autonomous func9ons, we believe that our in vivo findings provide substan9al evidence of the importance of LSD1 in skeletal muscle func9onality.It also captures the complex nature of physiological responses, which oeen gets diluted in isolated cell cultures.9. Numerous cri9cal concerns exist with the immunoprecipita9on experiments in the paper: a.The authors have used 10 % input, and most of the immunoprecipitated lanes have stronger bands than the input lane, which is inappropriate and impossible.For IP experiments, 10% of the protein extract were used for inputs, and the 90% lee were immunoprecipitated.If the efficiency of the IP is of 100%, the band would be 9 9mes stronger than that of the input.In our case, the quan9fica9on reveals that we never exceed 3 9mes the intensity of the input.Please find here various ar9cles with similar observa9ons: For LSD1: DOI: 10.1093/nar/gks031, 10.1038/s41388-021-02123-7, 10.7554/eLife.84618For GR: DOI:10.4049/jimmunol.1201776,10.3390/ijms24087130, 10.1093/nar/gkab226 b.Fig. 1B, 2D, 2F, 3A, 5F: It is unexpected to observe greater expression of the interac9ng partner protein than the immunoprecipitated protein.
We apologize for this lack of precision.From 200 ug of protein extracts IPed with LSD1 an9body 30 ul of proteins were loaded for GR detec9on and 10 ul for LSD1 detec9on, and vice versa for GR IP.This allows to avoid that the signal from the IP masks that of the co-IP.This procedure has been used for the various panels.This point is now stated in the method sec9on (page 27, lines 630-633).c.Also, the authors have used single IgG control for two independent immunoprecipita9on reac9ons performed with two different an9bodies, which may also be wrong.Please confirm if the origin of the secondary an9bodies against LSD1 and GR primary an9bodies is the same.Please verify the results and add loading controls to the panels.Rabbit IgG has been used as been used as control since NRF1, LSD1 and GR an9bodies were generated in this species (page 27, lines 629).In general, loading controls are not presented for IP experiments as they are not informa9ve (please see abovemen9oned ar9cles among others).d.Note that, oddly, the input bands in the top and bo^om panels are non-iden9cal in Fig. 1B.As men9oned in point 9b, for two-direc9ons IP experiments, the IPed material was split in two and loaded on two different gels since the molecular weight of GR and LSD1 is too close, and striping the membrane might result in signal lowering.e.In Fig. 4G: Are the IgG an9bodies used for Trim63 and Ddit4 of different origins?
There was no Fig.4g in the original version of the manuscript.However, in Fig. 5g, ChIP-qPCR was performed with an9-GR and an9-LSD1 an9bodies produced in rabbit.Thus, IgGs from that species were used for the analysis of the two loci presented.10.Cri9cal controls are missing in the figures.In Figure 6, (A-E), extended figures 6 and 7B-the CC-90011 alone treatment group is missing.Without this group, it would be hard to interpret the data shown in Figures 6, extended Figures 6 and 7. Our primary aim was to determine the implica9ons of LSD1 modula9on on DEX-induced muscle atrophy.The design of our experiments and controls was intended to dis9nguish the combined effects of the inhibitor and DEX.To determine the independent effects of the LSD1 inhibitor on GR ac9vity, CC-90011 control has now been included in the various panels of new Fig. 6, 7, S7. 11.Fig. 1G-I: Given that GR is a major transcrip9on factor for atrogenes, it is crucial to reveal whether LSD1 interacts with the atrogene loci in myofibres at physiological GC levels.To address this point, two examples, namely Fbxo32 (Atrogin1) and Trim63 (Murf1) loci, were provided in Fig. S1f in the original version of the manuscript.We now provide addi9onal examples in new Fig. and S2g.
12. Fig. 6A, B: It is difficult to interpret the effect of LSD on DEX-induced muscle atrophy without first ascertaining the impact of the LSD1 inhibitor itself on GR ac9vity.It is strongly recommended to validate the inhibitor in the mouse model used.We appreciate the construc9ve feedback on the interpreta9on of the effects of LSD1 on DEX-induced muscle atrophy in original Fig. 6a and 6b (new Fig. 7a, b), and implemented the figure as men9oned in point 10.Of note, the effect of CC-90011 on GR ac9vity was assessed by ChIP-qPCR as CC-90011 decreases GR binding to DNA (new Fig. 7c).
13. Since the primary outcome of the paper is to characterize the use of LSD1 inhibitor to circumvent the muscle atrophy phenotype associated with the use of DEXA as an an9-inflammatory drug, it is crucial to observe the cytokine and chemokine profile in DEX-treated control and LSD-/-mice, in addi9on to the immune cell popula9on shown in the manuscript.We agree with Reviewer 1 on the importance of the impact of the LSD1 inhibitor on DEX-associated muscle atrophy in parallel with GR an9-inflammatory ac9vi9es.To this aim, we provide two addi9onal lines of evidence.Using naive Th0 lymphocytes or Th17 differen9ated cells, we now show that whereas a 24h DEX treatment at 100 nM decreases IL17 produc9on by more than 3 folds, a 24h CC-90011 administra9on at 100 nM had no effect on interleukin produc9on, and did not impact DEX-dependent decrease in IL17 levels (new Fig. 7f).Moreover, the analysis of an experimental model of inflammatory bowel disease shows that LSD1 inhibi9on does not affect the an9-inflammatory effects of DEX on colon, but protects fast-twitch muscles from atrophy (new Fig. 8).
14. Fig. S1H-I: Please assess the extent and 9ssue specificity of LSD1 deple9on in the mouse model by western bloqng.The authors may test the expression of LSD1 in different muscles and organs.We now added the western blot characteriza9on of LSD1 protein levels in gastrocnemius, soleus, 9bialis and quadriceps muscles, as well as in epidydimal white adipose 9ssue and spleen (new Fig. S1i  and S3a).Moreover, immunofluorescent detec9on of LSD1 clearly shows that LSD1 is expressed in myofiber nuclei of control mice but not in those of LSD1 skm-/-and LSD1 (i)skm-/-mice (Fig. S1j and S3b).15.Fig. 3: To assess the role of the GR/LSD1 complex in muscle atrophy, it is impera9ve to evaluate the ac9vity of GR under starva9on condi9ons in control and LSDskm-/-mice.As men9oned in point 5, GR ac9vity is s9mulated under starva9on condi9ons in WT mice, as shown by increased transcript levels of classical GR target genes such as Atrogin1, Murf1 and Ddit4 (Fig. 3h and S3l), associated with GR binding at these loci (Fig. 4e).Moreover, the data show that the phenotype of LSD1 (i)skm-/-mice parallels that of GR (i)skm-/-mice in our experimental seqngs.Notably, the atrogenes are much less induced by starva9on in both LSD1 (i)skm-/-and GR (i)skm-/-mice.This observa9on strengthens our hypothesis regarding the interplay between LSD1 and GR in regula9ng muscle response to starva9on.
16. Fig. S4F: Given that FAD is deple9on upon starva9on, the enzyma9c ac9vity of LSD1 may be expected to reduce upon food depriva9on.It is, therefore, important to inves9gate and comment upon the 9me-sensi9ve role of LSD1 in GR-mediated ac9va9on of starva9on-induced muscle atrophy.We agree with the referee and implemented this point in the discussion, paralleling the data obtained in Fig. S4f with the ChIP-qPCR kine9cs shown in Fig. 4e.This point was discussed in the original version of the discussion of the manuscript (page 14 lines 325-331, now page 19 lines 438-443).17.Fig. S1J, 3A, 3E, 4C: Throughout the paper, authors have characterized LSD func9on almost exclusively in the gastrocnemius muscle.Verifying these cri9cal results in other important muscle types, especially the 9bialis anterior (among other Type I and Type II muscle fibers), would be interes9ng.We used gastrocnemius as it is a glucocor9coid-sensi9ve fast-twitch muscle composed of various type-2 fibers.Nonetheless, our data provide evidence that all the analyzed fast muscles (gastrocnemius, 9bialis and quadriceps) are similarly affected by either DEX or starva9on, as shown by decreased muscle mass, contrary to soleus muscle (Fig. 3b-d, S3d, f, g, 5a and S5a).As further evidence, we now provide western blot analysis of anabolic and catabolic pathway ac9vi9es in quadriceps muscle (Fig. 3f, g, S3j-l, 5d, e and S5d-f).In addi9on, GR and LSD1 co-localiza9on is now presented in the various muscles (new Fig. S1b).
18. Fig. 3E: Electron microscopy images are required to be quan9fied.Also, these results need to be verified by other methods.The measure of muscle weight and CSA, as well as RT-PCR analysis on genes from the proteasome system and western blot analysis of actors of the protein degrada9on pathway provide quan9ta9ve datasets for muscle atrophy.We presented electron microscopy pictures to characterize myofibers' defects.Moreover, we men9oned in the original version of the manuscript that "ultrastructural analysis of gastrocnemius muscles revealed disrup9ons of myofibrils in more than 60% of the sarcomeres aeer starva9on of control mice, with loss of myofilaments, rupture of Z-lines and enlarged sarcoplasm, whereas less than 20% of the sarcomeres were damaged in muscles of LSD1 (i)skm-/-and GR (i)skm-/-mice".Note however that electron microscopy is a semi-quan9ta9ve method and should be considered as addi9onal evidence for the phenotype.
19. Fig. 5D, E: Inconsistent use of loading controls in western bloqng.To be consistent, GAPDH western blot has been replaced by that of Tubulin in Fig. 5d (new Fig. 5f).Both are conserved in the source data file.20.Fig. 4C, 5D-E: The authors have not quan9fied the western blots in this manuscript.All western blots need to be quan9fied and presented.Protein quan9fica9on was already provided in Fig. 4d for Fig. 4c.Quan9fica9on has now been added next to the addi9onal western blot panels.3C-fiber cross-sec9on area (CSA) of GR(i)skm-/-mice starved for 48 h is much higher than the GR(i)skm-/-mice fed animals, which I feel is impossible.Fig. S3C: This figure indicates that starva9on induces hypertrophy in the muscles of GR-/-mice.This is highly improbable since starva9on is well known to inhibit protein synthesis.Furthermore, Fig. S3H indicates upregula9on in mRNA expression of atrogenes, contradic9ng the hypertrophy phenotype reported.

Extended figure
Original Fig. S3d (new Fig. S3g) shows no sta9s9cal difference in the average CSA of GR (i)skm-/-fed and starved mice.Moreover, a 2-way ANOVA analysis has been added on new Fig.3c and S3f to show that there is no sta9s9cal difference in CSA distribu9on between these two condi9ons, even for fibers with an area >5000.The table recapitula9ng ANOVA's results is provided in the source data file.
Minor comments: 1. Please ensure uniform color coding across data figures in the manuscript.This point has been implemented.2. Fig. 1H, S1E: Since the study is focused on skeletal muscles, please avoid displaying cardiacassociated genes in the list to avoid confusion.In the various figures showing pathways analysis, we presented the various bioinforma9cs annota9ons obtained from the KEGG analysis.We did not select any in par9cular.Note that the one en9tled "MicroRNAs in Cardiomyocyte Hypertrophy" contains genes expressed in skeletal muscle.
3. Fig. 6, 7B: Please denote groups as Vehicle, DEX, and DEX+CC in the figure for simplicity.This point has been implemented.

Reviewer #2 (Remarks to the Author):
In this manuscript, Cai and colleagues inves9gate the molecular details underlying glucocor9coidinduced skeletal muscle atrophy, which limits the use of synthe9c GC in pa9ents that require chronic administra9ons.They report that LSD1 interacts with both GR and NRF1 to regulate gene expression.Pharmacological inhibitors of LSD1 a^enuate muscle atrophy, providing evidence for the involvement of LSD1 and therapeu9c promising applica9ons.The number of reports that establish a role for LSD1 in skeletal muscle is increasing, so is the 9ght rela9onship with steroid hormone receptors.The manuscript is very well wri^en, results are clear and conclusions are appropriate.Before publica9on, we have the following sugges9ons that may improve to strength the authors' conclusions.We thank the reviewer for his/her very posi9ve feedback on our manuscript.His/her construc9ve sugges9ons will further improve the quality of our work.1) In Fig. 1 the authors show colocaliza9on of LSD1 and GR but not all the nuclei are posi9ve, can the author stain for the different fiber types with MyHC marker subtype to establish whether there is a preferen9al expression of type IIb vs intermediate or oxida9ve fibers?Is there a homogeneous distribu9on of double posi9ve nuclei in the en9re transversal sec9on, as the fiber type composi9on of gastrocnemius is not uniform.We performed a co-staining with GR (mouse IgG1) /LSD1 (rabbit) in slow-, fast-and mixed muscles, but could not co-stain with MyHC as all these an9bodies were produced in mouse.Our data show that GR and LSD1 evenly colocalize in the various analyzed muscles.Note that slow-twitch fibers are usually auto-fluoresce and are thus visible in green on our images (new Fig. S1b).Thus, even though GR and LSD1 co-localize in the different types of fibers, their ac9on on muscle atrophy is specific for the fasttwitch ones, indica9ng that addi9onal factors are selec9vely involved in fast/mixed-twitched fibers to induce atrophy.This point is now men9oned in the discussion (page 17 lines 399-402).
2) In the IP experiment, the lower band of LSD1, that also the authors no9ce, is interes9ng, could that be another isoform (alterna9ve splicing) or another member of the KDM family that interacts with GR? IP with an an9-GR Ab pulls down a lower band that is not visible in the input, may be because it is not enriched enough, but the upper isoform is not visible.This is problema9c.However, CHIP analysis shows interac9on at the chroma9n level.But the authors should clarify why the bands have different size, or reconsider the interpreta9on of the IP experiments.LSD1 has isoforms (2a, 8a, etc).We appreciate the opportunity to clarify this aspect of our study.LSD1 has a molecular weight of around 93 kDa and usually migrates between 110 and 120 kDa.In skeletal muscle, two isoforms coexist, the nLSD1 and the LSD1 2a that migrates 5 kDa upper than nLSD1 (Fig. R3A).The presence of these two isoforms was confirmed by Sashimi plot analysis obtained from RNA-seq datasets of gastrocnemius muscle (Fig. R3B).Note that the two bands are not always visible according to the percentage of the gel or the mount of protein loaded.Upon immunoprecipita9on with GR or NRF1 an9bodies, and LSD1 to a less extent, LSD1 was detected at a molecular weight between 100 and 110 kDa, which was shown by using both N-and C-terminal LSD1 an9bodies (Fig. R3C).Since similar data were obtained with an9-GR and an9-NRF1 an9bodies, we did not ques9on the an9body use for IP.We then ques9oned whether this issue was a problem of species since LSD1 an9bodies were originally designed for human usage.Thus, we tested LSD1/GR interac9on in LHCNM2 cells, but evenly obtained the band between 100 and 110 kDa upon GR IP (Fig. 6b).Aeer unsuccessfully tes9ng various post-transla9onal modifica9ons, we thus suspected a technical issue.Aeer tes9ng various parameters, we found that the observed molecular weight discrepancy originates from LSD1 sensi9vity to salt and/or pH composi9on of the elu9on buffer.In summary, the samples obtained from IP contain concentra9on of salt, ion chela9ng agents and pH values that differ from the input samples, and can affect the migra9on rate of the LSD1 protein within the acrylamide gel.Adjus9ng this parameter by adding for instance RIPA buffer to the IP samples mi9gates this issue (Fig. R3D).3544) and C-terminal (20752) an4-LSD1 an4bodies.IgG served as a control for immunoprecipita4on.D. Representa4ve western blot analysis of gastrocnemius muscle nuclear extracts from control (Ctrl) and LSD1 skm-/-mice immunoprecipitated with an4-GR or an4-LSD1 an4bodies and eluted in the presence of lysis buffer.Membranes were decorated with an4-LSD1 an4bodies.IgG served as a control for immunoprecipita4on.
3) Were the CHIP on target valida9ons performed on different samples as those used for CHIPseq?If so, this strengthens the valida9on analysis.ChIP-seq and ChIP-qPCR analyses were performed on independent cohorts.This point has been implemented in the method sec9on of the revised manuscript (page 28 line 674).
4) The first sentence of the figure cap9ons may be more informa9ve with respect to "Characteriza9on of…", "Role of…" can it be a sentence summarizing the main message of the figure?As proposed by the referee, the cap9ons have been modified.5) Is the interac9on of GR with LSD1 and NRF1 modulated by ligand?Can this be tested in cells treated with vehicle or GC? New Fig. 5g now provides evidence that the interac9on of GR with LSD1 and NRF1 is not affected modulated by pharmacological levels of DEX.However, circula9ng glucocor9coids are present in endogenous condi9ons.Thus, in addi9on to these data, we performed coIP experiments in LHCNM2 cells grown for 24h with charcoal-treated or in full medium (Fig. R4 and new Fig.6b).The decrease in the interac9on in charcoal-treated cells indicates that the interac9on is ligand-dependent.
6) The observa9on that LSD1 is required for interac9on of GR with NRF1 (Fig 2F) suggests a specific effect, is it possible that LSD1 demethylates GR? Or is this effect (fig 2G) occurring at the chroma9n level?To thoroughly address the ques9on of LSD1 demethylase ac9vity on GR, we will combine this answer with that of point 9 (see hereaeer).7) I suggest to move to main figure 3 analysis of CSA in control and starved mice with the rela9ve quan9fica9on.Rela9ve to autophagy, WB analysis of p62 and LC3 I/II should be added to conclude that autophagy is not induced and to determine is the autophagy flux is regular.As proposed by the referee, the analysis of CSA in control and starved mice has been moved to main figure 3 and we performed western blot analysis of p62 and LC3 I/II, the expression of which is similar between control and LSD1 mutant mice (Fig. 3f, g and 5d, e).
8) What would be the benefit to use CC-90011 with respect to TCP, for instance?Tranylcypromine is a cell-permeable phenylcyclopropylamine that inhibits the monoamine oxidase and histone demethylase ac9vi9es, respec9vely, of MAO A/B (Ki = 101.9and 16.0 M, respec9vely) and LSD1/2 (Ki = 242.7 and 180.0 M, respec9vely), four members of a flavin-dependent amine oxidase family enzymes, by a covalent adduct forma9on with the enzyme-bound FAD, and is therefore not specific for LSD1.Tranylcypromine irreversibly inhibits LSD1 with an IC50 value of 20.7 µM.Pulrodemstat (CC-90011) is a potent, selec9ve, reversible and orally ac9ve inhibitor of lysine specific demethylase-1 (LSD1) with an IC50 of 0.25 nM, which jus9fied our choice.A sentence clarifying this point has been added in the discussion (page 19 lines 444-449).
9) The effect of the inhibitor (occurring through the cataly9c ac9vity) implies that LSD1 demethylase ac9vity is important, is it only involved in histone modifica9on or is it the result of direct modifica9on of GR or NRF1?LSD1 has been shown to demethylate the non-histone protein P53 (DOI: 10.1038/nature06092).To our knowledge, there has been no study describing NRF1 and GR methyla9on (see Gene cards website and the following reviews: DOI: 10.1016/j.biopha.2023.115145 and 10.1074/jbc.272.30.18732).Therefore, we tested if these two factors are methylated.Using mouse muscles, we performed IP against GR and NRF1, followed by WB with pan-Methyl-Lysin an9body.Our results did not reveal GR or NRF1 methyla9on in muscle extracts (Fig. R5), thereby strongly indica9ng that LSD1 cannot further demethylate the two proteins.
Thus, we speculate that in our context LSD1 holds two main func9ons: the first one is to demethylate H3K9me1/2 to promote gene transcrip9on, and the second one is a scaffolding func9on to allow GR and NRF1 interac9on.NRF1 has previously been proposed to be a pioneer factor based on its ability to bind de novo hypomethylated DNA sites (h^ps://doi.org/10.1038/nbt.2798).NRF1 only bears canonical hallmarks of a pioneer factor in the absence of DNA methyla9on, where it can bind autonomously.These genomewide analyses thereby revealed that NRF1 occupies several thousand addi9onal sites in the unmethylated genome, resul9ng in increased transcrip9on.In a previous study, we showed that GR at skeletal muscle-specific enhancers interacts with NRF1 located at open-chroma9n promoter regions of target genes via the forma9on of chroma9n loops, to s9mulate gene transcrip9on.Fig 2 . of the current manuscript shows the molecular mechanism by which LSD1 mediates glucocor9coids' ac9on by allowing interac9on between GR at enhancers and NRF1 at promoters at physiological GC levels (see also Reviewer #1 points 2 and 8 and Reviewer #2 point 5), via a direct interac9on with the two transcrip9on factors.Pathway analysis revealed that the GR-LSD1-NRF1 complex is mainly bound to genes involved in protein degrada9on.We show in Fig. 5g that LSD1 is s9ll in complex with GR and NRF1 to promote target gene expression such as Murf1 and Trim63.Thus, NRF1 is not a regulator per se of muscle atrophy but is required for GR-dependent gene expression via LSD1 (new Fig. 2m and S2i).
11) The discussion and the manuscript would benefit if the authors take into considera9on recent findings that establish a role of LSD1 in muscle in the context of another steroid receptor, androgen receptor.Especially considering the anabolic effects of androgen signaling in muscle.LSD1 represses transcrip9on of several genes, but it transac9vates AR, is this the case for GR? Similar specific effect that boosts GR transac9va9on?Our data obtained from ChIP and RT-qPCR strongly indicate that LSD1 is required for GR-dependent transcrip9on.Similar to what has been shown for AR, luciferase assay at GR response element shows that LSD1 overexpression enhances GR ac9vity in the presence of DEX (Fig. R6), confirming that LSD1 acts as a co-ac9vator of GR.The discussion has been implemented accordingly (page 14 lines 372-376).
12) For sta9s9cal analysis, ANOVa (not t test) shall be used for more than 2 sample comparisons.T-test has been used for sta9s9cal analysis of Fig. 1f, in which each an9body was compared to its respec9ve IgG control.Since we did not aim comparing LSD1 with GR an9body, we did not perform ANOVA test.Fig. 2g has been reanalyzed with a One-way ANOVA with Tukey correc9on.

Reviewer #3 (Remarks to the Author):
The study by Cai and collaborators inves9gates the rela9onship between the lysine-specific demethylase LSD1 and the glucocor9coid receptor (GR) in the context of muscle atrophy.The Authors show that these molecules directly interact resul9ng in the modula9on of target gene expression.Among others, gene involved in the regula9on of muscle mass homeostasis are targeted by LSD1 and GR interac9on.The study is mainly based on gene9c approaches, also taking advantage of mice in which the expression of GR or LSD1 is down-regulated.The results showing the interac9on between LSD1 and GR are quite convincing, while few notes of care arise as for the relevance of such interac9on to muscle atrophy, which is inves9gated in mice exposed to fas9ng or administered dexamethasone (DEX).Specifically, the following issues should be taken into considera9on: We thank the reviewer for his/her construc9ve comments.We have implemented the suggested modifica9ons.
1) line 85-86: while it is conceivable that some GRs are located in the nucleus also in physiological condi9ons such as those analyzed by the Authors, Figure 1A shows no or very li^le cytoplasmic GR, while this la^er should be its main loca9on in this experimental seqng; Regarding GR localiza9on in Fig. 1a, it is important to note that, even in physiological condi9ons, circula9ng GC levels can induce GR transloca9on to the nucleus as shown previously (DOI: 10.1093/nar/gkab226).Please find addi9onal GR staining in mouse prostate in which GR is also essen9ally nuclear (Fig. R7).2) one of the main findings reported in the present study is that muscle-specific lack of LSD1 confers a significant resistance to was9ng induced in mice (age?ini9al body weight?) by 48 h fas9ng.Such a pa^ern is associated with inhibi9on of the expression of genes involved in catabolic pathways such as muscle-specific ubiqui9n ligases and autophagy and with preserva9on of muscle ultrastructure.However, these results do not rule out that other pathways in addi9on to the GR-dependent one(s) could be altered by the lack of LSD1 in the skeletal muscle.In this regard, 48 h fas9ng results in a severe substrate shortage.While adipose 9ssue reduc9on, more marked in LDS1-/-mice than in controls, likely a^empts to compensate such a shortage, it is hardly able to achieve a total 'buffer' against the lack of nutrients; it would have been interes9ng to see the effects of LSD1 inhibitor in fasted animals; We thank the reviewer for the interes9ng comment.As requested, the ini9al body weight of the mice used for the starva9on experiment is now shown in Fig. 3b and S3d.In addi9on, the age at which starva9on was performed is now stated in the Methods sec9on (page 21 line 489) and in the figure legends.As men9oned in the manuscript, is not a good model to treat with an LSD1 inhibitor as LSD1 is released from the chroma9n 6h aeer food depriva9on.Moreover, such an experiment has li^le clinical relevance.
3) the second main observa9on of the study is that LSD1 down-regula9on protects mice against DEXinduced atrophy, apparently without affec9ng DEX an9-inflammatory proper9es.However, only indirect evidence is provided in this regard, showing that spleen changes in size and both spleen resident and circula9ng immune cell sub-popula9ons do not differ in the presence or in the absence of the LSD1 inhibitor.In this regard, the lack of effect on the an9-inflammatory proper9es should be demonstrated using the LSD1 inhibitor on models such as the experimental autoimmune encephalomyeli9s or inflammatory bowel disease; As proposed by the referee, we used an experimental model of inflammatory bowel disease.Our data provide evidence that LSD1 inhibi9on under these experimental condi9ons also does not affect the an9-inflammatory effects of DEX, and par9ally protects fast-twitch muscle from atrophy (new Fig. 8, and result sec9on pages 14-15).4) control mice treated with the LSD1 inhibitor are not included in the experimental design, while they should; As proposed by the referee, the CC-90011 control has been included in new Fig. 6, S6 and 7. 5) LSD1 lack of ac9on on the an9-inflammatory ac9vity does not really fit with data reported in Figure 1I, which shows an up-regula9on of genes involved in the Inflammatory Response Pathway; It is correct that Fig. 1i shows that LSD1 and GR are involved in the transcrip9onal regula9on of genes involved in the Inflammatory Response Pathway in skeletal muscle.However, this does not imply that they will carry out a similar func9on in immune cells or in spleen.Indeed, whereas GR transcrip9onal ac9vity is mainly driven by its response element (GRE) in myofibers, it is thought to act via the transrepression of transcrip9on factors such as NFkB, AP1 or STAT3 to control the immune response.A sentence to discuss this point has been added in the discussion (page 19 lines 457-461).6) most of the results obtained in starved or DEX-exposed mice refer to gene expression data.However, since changes of mRNA levels do not necessarily match with protein expression and ac9vity, at least some of the markers of protein hypercatabolism should be assessed at the protein level; As proposed by the referee, the western blot analysis of genes involved in the AKT/mTOR/FOXO pathway has been included, confirming that GR (i)skm-/-and LSD1 (i)skm-/-mice are resistant to fas9ng-and DEX-induced protein catabolism (Fig. 3f, g, S3i-k, 5d, e and S5d-f).7) data on human myotubes do not add any par9cular cue, they could be cited as supplemental; We used human myotubes as a confirma9on of LSD1 cell-autonomous effects.We now provide addi9onal evidence on the forma9on of the GR/LSD1 complex in new Fig.6b.We implemented this dataset with primary Th0 and Th17 cells to modelize the effects of DEX in combina9on with CC-90011 on inflamma9on (new Fig. 7f).8) it is not correct to talk about 'muscle catabolism' or 'muscle degrada9on'.Indeed, a 9ssue per se cannot be degraded or synthe9zed, rather its components (protein, lipids, etc.) are processed during anabolic/catabolic reac9ons.These points have been implemented."Muscle catabolism" has been modified by muscle was9ng (Page 2 line 32), muscle atrophy (Page 4 line 74), muscle was9ng (Page 17 line 449), and "muscle degrada9on" by muscle was9ng (Page 10 line 234).Similarly, it is not correct to talk about muscle atrophy when referring to an in vitro system (line 56); We modified "muscle atrophy" by "protein degrada9on" (Page 12 line 299).9) finally, in lines 203-204 LSD1 is reported to play a role in 'ini9a9ng the food depriva9on process', which is a non-sense, since such a process starts when mice are no more allowed to reach food.We apologize for this lack of precision and modified the sentence now page 10 line 246 in the new version of the manuscript.

Figure R2 :Figure R1 :
Figure R2: GSEA analysis of genes differen4ally expressed in gastrocnemius muscle of 9-week-old control and LSD1 skm-/-mice.Pathways related to upregulated genes are presented in orange, when those related to down-regulated genes are in blue.

Figure R3 :
Figure R3: A. Western blot analysis of LSD1 levels in gastrocnemius muscles of wild-type mice on a 8% gel.B. Sashimi plot for alterna4vely spliced exon and flanking exons in four wild-type samples.Per-base expression is plo\ed on y-axis of Sashimi plot, genomic coordinates on x-axis, and mRNA isoforms quan4fied are shown on bo\om (exons in blue, introns as lines with arrow heads).nLSD1 and LSD1 2a isoforms are shown for each sample.C. Representa4ve western blot analysis of gastrocnemius muscle nuclear extracts from wild-type mice immunoprecipitated with an4-GR or an4-LSD1 an4bodies.Membranes were decorated with N-terminal (3544) and C-terminal (20752) an4-LSD1 an4bodies.IgG served as a control for immunoprecipita4on.D. Representa4ve western blot analysis of gastrocnemius muscle nuclear extracts from control (Ctrl) and LSD1 skm-/-mice immunoprecipitated with an4-GR or an4-LSD1 an4bodies and eluted in the presence of lysis buffer.Membranes were decorated with an4-LSD1 an4bodies.IgG served as a control for immunoprecipita4on.

Figure R4 :
Figure R4: Representa4ve western blot analysis of nuclear or cytoplasmic extracts of LHCN-M2 cells grown in full-medium or charcoal-treated medium, immunoprecipitated with an4-GR or an4-LSD1an4bodies.Rabbit IgG served as a control for immunoprecipita4on.
10) And what is the involvement of NRF1 in muscle atrophy?What do the data shown in Fig 2 add to the rest of the story?

Figure
Figure R6:Transac4va4on analysis in LHCNM2 myofibers transfected with a pGL3-promoter vector containing the 5'-GGAACAGAACACGGTGTAGCTGGGA-3' GR response element, 100 ng of a pSG5-GR expression vector, and 37.5 ng of a pSG5-LSD1 expression vector, in the presence or absence of 10 nM DEX.Data were normalized to Firefly expression.

Figure R7 :
Figure R7: Immunofluorescent detec4on of GR (red) in mouse prostate and skeletal muscle of 10-week-old mice.Nuclei are stained with DAPI (blue).